We aim to formulate rational general strategies for the specific inhibition of selected steroidogenic hydroxylation enzymes, as a component of therapeutic approaches to certain endocrine disorder including steroid dependent tumors. We request support for the execution of a stepwise plan for obtaining a detailed picture of the active site structure and reaction mechanism of the cholesterol side-chain cleavage enzyme, P450scc, which catalyses the rate-limiting initial step of steroid hormone biosynthesis. This proposal is based on our previous work, including development of mechanism-based inhibitors of P450scc, as well as our demonstration that electron spin echo spectroscopy (ESEEM) can measure interatomic distances in enzyme-substrate complexes of P450scc. We propose to (1) label and identify active site residues in the P450 peptide sequence with suicide substrates, and test mechanistic proposals with radical clock substrates and isotope incorporation experiments, (2) prepare aminosteroids as diagnostic ligands of specific P450's, (3) use deuterated steroids to measure distances from steroid atoms to the heme Fe of P450scc with ESEEM, (4) measure the S or Se to Fe distance in thia (selena) steroid-P450scc intermediates using EXAFS, (5) fit the sequence of P450scc to the structure of the partially homologous P450cam crystal structure in attempts to understand the derivatization and spectroscopic results, (6) test our understanding of mechanism by mutagenizing specific residues, (7) attempt to form and crystallize a robust P450scc-inhibitor complex for x-ray diffraction studies. Longer term we will extend this paradigm to other P450's, eg. the 11beta, 18, 19, and 17,20 hydroxylases. Support is also requested for refinement of a ferredoxin structure, solved in the previous period of this project.